Genome-wide mapping of nucleosome positioning and DNA methylation within individual DNA molecules

Abstract

DNA methylation and nucleosome positioning work together to generate chromatin structures that regulate gene expression. Nucleosomes are typically mapped using nuclease digestion requiring significant amounts of material and varying enzyme concentrations. We have developed a method (NOMe-seq) that uses a GpC methyltransferase (M.CviPI) and next generation sequencing to generate a high resolution footprint of nucleosome positioning genome-wide using less than 1 million cells while retaining endogenous DNA methylation information from the same DNA strand. Using a novel bioinformatics pipeline, we show a striking anti-correlation between nucleosome occupancy and DNA methylation at CTCF regions that is not present at promoters. We further show that the extent of nucleosome depletion at promoters is directly correlated to expression level and can accommodate multiple nucleosomes and provide genome-wide evidence that expressed non-CpG island promoters are nucleosome-depleted. Importantly, NOMe-seq obtains DNA methylation and nucleosome positioning information from the same DNA molecule, giving the first genome-wide DNA methylation and nucleosome positioning correlation at the single molecule, and thus, single cell level, that can be used to monitor disease progression and response to therapy.

Figure 1.

NOMe-seq can footprint a variety of chromatin structures. (A) After IMR90 cell nuclei are treated with M.CviPI, DNA is extracted, bisulfite-converted, and sequencing is performed. DNA methylation status is obtained from CpG dinucleotides, and nucleosome occupancy information is gained from the inaccessibility of the M.CviPI methyltransferase to GpC dinucleotides. The combination of DNA methylation and nucleosome occupancy data can reveal four distinct chromatin signatures: unmethylated and nucleosome-depleted, unmethylated and nucleosome-occupied, methylated and nucleosome-occupied, and methylated and nucleosome-depleted. (Black circles) Methylated CpG sites; (teal circles) accessible (methylated) GpC sites. (B) We found that 200 units of M.CviPI for 7.5 min followed by a boost of 100 units accurately revealed an NDR upstream of the TSS of HSPA5 (also known as GRP78), an active CGI promoter, while also showing that the polycomb repressed MYOD1 CGI promoter and methylation-silenced CpG-poor LAMB3 promoter were occupied by nucleosomes and inaccessible to M.CviPI, as expected. M.CviPI-inaccessible regions greater than 146 bp are covered by a pink rectangle indicating nucleosome occupancy. PCR amplicon sizes: HSPA5–447 bp, MYOD–474 bp, and LAMB3–426 bp.

Figure 2.

NOMe-seq diplays nucleosome occupancy profiles at specific loci and globally. (A) Broad view of the ATM promoter using a newly developed module of the IGV viewer (Thorvaldsdottir et al. 2012) to visualize NOMe-seq BAM alignment files. The top two tracks indicate endogenous DNA methylation (at HCG sites) in each of two GBM samples, while tracks 5 and 6 indicate GCH accessibility of the same GBM samples. (Red) Methylated sites (for both HCG and GCH); (blue) unmethylated sites (for both HCG and GCH). The promoters of ATM and NFAT are unmethylated (blue in top two tracks) and nucleosome-depleted (i.e., accessible and therefore methylated, and thus red in tracks 5 and 6). The same methylation and nucleosome occupancy pattern is seen for both GBM samples. Tracks 3 and 4 show average methylation levels derived from these tracks—at each individual HCG, the number of reads methylated at that HCG is divided by the total number of reads methylated and unmethylated. Average GCH methylation in tracks 7 and 8 is calculated as before but inverted (1-GCH) to indicate nucleosome protection as used throughout the main figures. The tool and source code are publicly available for download at the IGV project website: http://www.broadinstitute.org/igv/. (B,C) NOMe-seq reads were aligned to CTCF (B) and TSSs (C). Nucleosome positioning in IMR90 cells is indicated on the y-axis by inaccessibility to M.CviPI (1-GpC methylation; teal line) and the number of MNase sequencing reads (blue line). For MNase-seq, reads were aligned to 8709 CTCF sites, while 8687 CTCF sites had at least one GpC site that was covered by a minimum of three reads (B). For TSS, 42,103 promoters were used for MNase-seq, and 41,292 promoters had at least one GpC site that was covered by a minimum of three reads. (D,E) Gene promoters were divided into quartiles based on transcription level (Hawkins et al. 2010), and the corresponding M.CviPI inaccessibility (1-GCH, teal line) is plotted on the y-axis. (D) CpG island promoters. (E) Non-CpG island promoters. The NDR is stronger in more highly expressed genes and, in some cases, can be several hundred bp long to accommodate multiple nucleosomes.

Figure 3.

NOMe-seq reveals distinct chromatin configurations at CTCF sites and associated with specific histone modifications and promoter types. (A) NOMe-seq demonstrates unmethylated NDRs at CTCF sites in IMR90 and GBM cells, which are marked by a peak in inaccessibility at the CTCF site itself. Well-positioned nucleosomes flank CTCF sites, with DNA methylation peaking in between nucleosomes. 0 indicates the middle of the CTCF binding motif. CTCF binding sites were obtained from GSM935404. (B) NOMe-seq distinguishes the three major promoter states at promoters in IMR90 cells—active H3K4me3-marked promoters are unmethylated and contain a NDR upstream and well-positioned nucleosomes after the TSS. TSSs are indicated on the x-axes as 0. Repressed/poised H3K27me3-marked promoters are unmethylated and nucleosome-occupied. Methylated promoters are nucleosome-occupied. The y-axis indicates M.CviPI inaccessibility (1-CpG; teal) and CpG methylation level. (C) In IMR90 cells, CpG island promoters are characterized by a lack of CpG methylation, an upstream NDR, and well-positioned nucleosomes after the TSS. The majority of CpG island promoters are unmethylated (11,165) and display the same pattern, while methylated CpG island promoters (781) are nucleosome-occupied and inaccessible to M.CviPI. (D) Non-CpG island promoters are generally characterized by CpG methylation and inaccessibility to M.CviPI, indicating nucleosome occupancy. The few unmethylated non-CpG island promoters (1397) are depleted of nucleosomes upstream of the TSS, while the majority of non-CpG island promoters (4668) are nucleosome-occupied and inaccessible to M.CviPI. M.CviPI inaccessibility is plotted (1-GCH) in teal and CpG methylation (CGH) in black.

Figure 4.

Combinatorial epigenomic signatures reveal functional chromatin. (A–C) Nucleosome occupancy levels (“percent of GpCs protected”) are shown stratified by the methylation status of nearby CpGs (within 20 bp). For each element type, this analysis was performed twice—once sampling randomly across all reads covering the same genomic position as the GpC (left plots, labeled “across all reads”) and a second time using only the methylation status from the same read (right plots, labeled “on same read”) (see Methods). All three examples show nucleosome depletion associated primarily with the unmethylated state, but while predicted AP-1 binding motifs (A) display this in both population and within-read profiles, enhancers and promoters marked by the opposing K4me3 and meC (B,C) show this association only in the within-read analysis. 0 refers to the center of the AP-1 binding motif (A), the peak of DNase HS within K4me1-marked regions (B) and TSSs (C). (D) Search strategy for finding divergent chromatin alleles (DCA) by searching TSS regions for at least two reads with opposing chromatin profiles in IMR90 cells. (E) Promoters that exist in both nucleosome-depleted and unmethylated and nucleosome-occupied and methylated are enriched on the X chromosome. Seven hundred and forty-two DCA genes were compared to randomized sets of 742 genes—1000 trials were performed and the standard deviation is shown for the number on each chromosome. A P-value was determined from the X chromosome using a binomial test with the probability determined by the random trials. (F) DCA genes were compared to 1000 randomized gene sets for the number within 50 kb of known imprinted genes.

Figure 5.

Validation of DCA promoters. PCR amplicons were cloned, and several colonies were sequenced to visualize two distinct chromatin configurations of an imprinted gene, SNRPN (A), and an X-linked locus, DLG3 (B), and a novelly identified DCA gene, ZNF597 (C). (Black) DNA methylation of CpG sites; (teal) GpC accessibility. Pink bars indicate nucleosome positioning.